Microelectronic sensor device for detecting label particles

ABSTRACT

The invention relates to a microelectronic sensor device for the detection of target components that comprise label particles, for example magnetic particles ( 1 ). The sensor device comprises a carrier ( 11 ) with a binding surface ( 12 ) at which target components can collect and optionally bind to specific capture elements. An input light beam (L 1 ) is transmitted into the carrier and totally internally reflected at the binding surface ( 12 ). The amount of light in the output light beam (L 2 ) and optionally also of fluorescence light emitted by target components at the binding surface is then detected by a light detector ( 31 ). Evanescent light generated during the total internal reflection is affected (absorbed, scattered) by target components and/or label particles ( 1 ) at the binding surface ( 12 ) and will therefore be missing in the output light beam (L 2 ). This can be used to determine the amount of target components at the binding surface ( 12 ) from the amount of light in the output light beam (L 2 , L 2   a , L 2   b ). A magnetic field generator ( 41 ) is optionally used to generate a magnetic field (B) at the binding surface ( 12 ) by which magnetic label particles ( 1 ) can be manipulated, for example 15 attracted or repelled.

The invention relates to a microelectronic sensor device and a methodfor the detection of target components, for example biologicalmolecules, comprising label particles. Moreover, it relates to a carrierand a well-plate that are particularly suited for such a sensor device.

The US 2005/0048599 A1 discloses a method for the investigation ofmicroorganisms that are tagged with particles such that a (e.g.magnetic) force can be exerted on them. In one embodiment of thismethod, a light beam is directed through a transparent material to asurface where it is totally internally reflected. Light of this beamthat leaves the transparent material as an evanescent wave is scatteredby microorganisms and/or other components at the surface and thendetected by a photodetector or used to illuminate the microorganisms forvisual observation.

Based on this situation it was an object of the present invention toprovide means for an improved detection of target components comprisinglabel particles. In particular, it is desired that the method is simpleand that its sensitivity and/or accuracy is improved with respect to thestate of the art.

This object is achieved by a microelectronic sensor device according toclaim 1, a carrier according to claim 19, a well-plate according toclaim 20, and a method according to claim 28. Preferred embodiments aredisclosed in the dependent claims.

The microelectronic sensor device according to the present inventionserves for the qualitative or quantitative detection of targetcomponents comprising label particles, wherein the target components mayfor example be biological substances like biomolecules, complexes, cellfractions or cells. The term “label particle” shall denote a particle(atom, molecule, complex, nanoparticle, microparticle etc.) that hassome property (e.g. optical density, magnetic susceptibility, electricalcharge, fluorescence, radioactivity, etc.) which can be detected, thusindirectly revealing the presence of the associated target component.The microelectronic sensor device comprises the following components:

-   -   a) A carrier with a binding surface at which target components        can collect. The term “binding surface” is chosen here primarily        as a unique reference to a particular part of the surface of the        carrier, and though the target components will in many        applications actually bind to said surface, this does not        necessarily need to be the case. All that is required is that        the target components can reach the binding surface to collect        there (typically in concentrations determined by parameters        associated to the target components, to their interaction with        the binding surface, to their mobility and the like). The        carrier should have a high transparency for light of a given        spectral range, particularly light emitted by the light source        that will be defined below. The carrier may for example be        produced from glass or some transparent plastic.    -   b) A light source for emitting a light beam, called “input light        beam” in the following, into the aforementioned carrier such        that it is totally internally reflected in an investigation        region at the binding surface of the carrier. The light source        may for example be a laser or a light emitting diode (LED),        optionally provided with some optics for shaping and directing        the input light beam. The “investigation region” may be a        sub-region of the binding surface or comprise the complete        binding surface; it will typically have the shape of a        substantially circular spot that is illuminated by the input        light beam. Moreover, it should be noted that the occurrence of        total internal reflection requires that the refractive index of        the carrier is larger than the refractive index of the material        adjacent to the binding surface. This is for example the case if        the carrier is made from glass (n=1.6) and the adjacent material        is water (n=1.3). It should further be noted that the term        “total internal reflection” shall include the case called        “frustrated total internal reflection”, where some of the        incident light is lost (absorbed, scattered etc.) during the        reflection process.    -   c) A light detector for determining the amount of light in an        “output light beam”, which comprises light that stems from the        aforementioned total internal reflection of the input light        beam. It is not necessary that the output light beam comprises        all the totally internally reflected light (though this will        preferably be the case), as some of this light may for example        be used for other purposes or simply be lost, or that it        completely consists of totally internally reflected light, as it        may also comprise e.g. scattered light or fluorescence light.

The detector may comprise any suitable sensor or plurality of sensors bywhich light of a given spectrum can be detected, for example aphotodiode, a photo resistor, a photocell, a CCD chip, or a photomultiplier tube.

The described microelectronic sensor device allows a sensitive andprecise quantitative or qualitative detection of target components in aninvestigation region at the binding surface. This is due to the factthat the totally internally reflected input light beam generates anevanescent wave that extends from the carrier surface a short distanceinto the adjacent material. If light of this evanescent wave isscattered or absorbed by label particles bound to target componentspresent at the binding surface, it will be missing in the output lightbeam. The amount of light in the output light beam (more precisely theamount of light missing in the output light beam when compared to theinput light beam) is therefore an indication of the presence and theamount of label particles at the binding surface. One advantage of thedescribed optical detection procedure comprises its accuracy as theevanescent waves explore only a small volume of typically 10 to 300 nmthickness directly above the binding surface, thus avoiding disturbancesfrom the bulk material behind this volume. A high sensitivity isachieved when the reflected light is measured as all effects aredetected that reduce the amount of totally internally reflected light.Moreover, the optical detection can optionally be performed from adistance, i.e. without mechanical contact between the carrier and thelight source or light detector.

The microelectronic sensor device may particularly be designed such thatthe totally internally reflected light beam becomes frustrated resultingin a decrease of the totally internally reflected light intensity whenthe label-particles that are bound to the target components aremacroscopic scattering and/or absorbing particles.

In a preferred embodiment of the invention, the microelectronic sensordevice comprises a field generator for generating a magnetic and/or anelectrical field that can affect the label particles. The fieldgenerator may for example be realized by a permanent magnet, a wire, apair of electrodes, or a coil. The generated field may affect the labelparticles for instance by inducing a magnetization or a polarizationand/or by exerting forces on them. Such a microelectronic sensor deviceallows a versatile manipulation of target components via fields, whichmay for example be used to accelerate the collection of targetcomponents at the binding surface and/or to remove undesired (unboundor, in a stringency test, weakly bound) components from the bindingsurface.

In the general case, the space next to the carrier at the side of thebinding surface may be arbitrarily designed. It is for example possiblethat this space is exterior to the microelectronic sensor device andthat target components are applied to the binding surface by spraying orpainting; the space may also be open to the surroundings for detectingtarget components in e.g. the ambient atmosphere. Moreover, it ispossible that the target components reach the binding surface throughthe carrier, e.g. by diffusion. In preferred embodiments of theinvention, the microelectronic sensor device comprises however a samplechamber which is located adjacent to the binding surface and in which asample with target components can be provided. The sample chamber istypically an empty cavity or a cavity filled with some substance like agel that may absorb a sample substance; it may be an open cavity, aclosed cavity, or a cavity connected to other cavities by fluidconnection channels.

As was already mentioned, the microelectronic sensor device may be usedfor a qualitative detection of target components, yielding for example asimple binary response with respect to a particular target molecule(“present” or “not-present”). Preferably the sensor device compriseshowever an evaluation module for quantitatively determining the amountof target components in the investigation region from the detectedoutput light beam. This can for example be based on the fact that theamount of light in an evanescent light wave, that is absorbed orscattered by label particles, is proportional to the concentration ofthe target components bound to the label particles in the investigationregion. The amount of target components in the investigation region mayin turn be indicative of the concentration of these components in anadjacent sample fluid according to the kinetics of the related bindingprocesses.

In a further development of the aforementioned embodiment, themicroelectronic sensor device comprises a recording module formonitoring the determined amount of light in the output light beam overan observation period. Thus it will be possible to monitor the kineticswith which target components collect at or depart from the bindingsurface. This may reveal valuable information about the targetcomponents and/or the prevailing ambient conditions. The evaluationmodule and/or the recording module are typically coupled to the lightdetector and may be realized by some data processing hardware, e.g. amicrocomputer, together with associated software.

Up to now the description of the microelectronic sensor device includedthe case that only a single investigation region is present on thebinding surface. In the following, several embodiments of themicroelectronic sensor device will be considered in which the carriercomprises a plurality of investigation regions at which different inputlight beams can be totally internally reflected. One carrier then allowsthe processing of several investigation regions and thus for example thesearch for different target components, the observation of the sametarget components under different conditions and/or the sampling ofseveral measurements for statistical purposes. The “different inputlight beams” may optionally be components of one broad light beam thatis homogeneously generated by the light source.

The different input light beams that are used in the aforementionedembodiment may be different with respect to time. This is for examplethe case if the microelectronic sensor device comprises a scanningmodule for sequentially coupling the light source to differentinvestigation regions. Alternatively or additionally, it may comprise ascanning module for optically coupling the light detector to differentinvestigation regions on the binding surface. The scanning modules mayfor example comprise optical components like lenses or mirrors fordirecting the incident or the output light beam in a suitable way. Thescanning modules may also comprise means for moving the carrier withrespect to the light source and/or light detector.

In another embodiment of the microelectronic sensor device with aplurality of investigation regions, a plurality of light sources and/ora plurality of light detectors is present that are directed to differentinvestigation regions at the binding surface. In this case it ispossible to process a plurality of investigation regions simultaneously,thus speeding-up the associated measurement process accordingly. Thisembodiment can of course be combined with the previous one, i.e. theremay for example be a scanning module for scanning the input light beamsof a plurality of light sources over different arrays of investigationregions and/or a scanning module for directing the output light beamsfrom different arrays of investigation regions to a plurality of lightdetectors. By using scanning modules, the number of lightsources/detectors can be kept smaller than the number of investigationregions.

In another embodiment with a plurality of investigation regions, themicroelectronic sensor device comprises a plurality of individuallycontrollable (magnetic or electrical) field generators that areassociated to different investigation regions. In this case it ispossible to manipulate the label particles in each investigation regionindividually according to the requirements of the particular tests thatshall be performed there.

The microelectronic sensor device may in principle be used with any kindof label particles. It is however preferably provided with labelparticles that specifically fit to the other components of the device.The sensor device may especially comprise label particles with a mantleof a transparent material, wherein this mantle typically covers(completely or partially) one or more kernels of another material, e.g.iron-oxide grains. In this case light of an evanescent light wave at thebinding surface can readily enter the label particles where it isabsorbed and/or scattered and thus lost for the output light beam. Thetransparent material of the mantle may particularly be a material with asimilar refractive index as the material of the carrier, because thisoptimizes the transition of light from the carrier to the labelparticles. The mantle may for example consist of the same material asthe carrier.

The microelectronic sensor device may optionally comprise a “secondlight detector” for determining (qualitatively or quantitatively)fluorescence light emitted by target components at the binding surface.The fluorescence can be stimulated by the evanescent wave of the inputlight beam in a small volume adjacent to the binding surface and then bedetected, thus indicating the presence (and amount) of fluorescenttarget components.

In another embodiment of the invention, the microelectronic sensordevice comprises an input-light monitoring sensor for determining theamount of light in the input light beam. This allows to take said amountinto consideration during a (quantitative) evaluation of themeasurements of the output light beam and/or to control the input lightbeam in a feedback loop.

The input-light monitoring sensor can be integrated into the lightsource, which provides a robust and compact design and which isfavorable for an integration in a feedback control loop. Alternatively,the input-light monitoring sensor (or at least a part of it) can bedisposed outside the light source as an independent component. Thelatter arrangement has the advantage that the measurement of this sensorcan be better focused on the actual input light beam as it enters thecarrier because the monitoring measurement takes place behind opticalelements like lenses or pinholes that are typically present in the lightpath of the light source.

It was already mentioned that the measurement results of the input-lightmonitoring sensor can be related to the amount of light in the outputlight beam that is determined by the light detector. The microelectronicsensor device may therefore comprise an evaluation module that isadapted to execute such a relation. To this end, the evaluation moduleis typically provided with signals from the input-light monitoringsensor and the light detector which represent the determined amounts oflight. The evaluation module may optionally preprocess these signals,e.g. (low-pass) filter them. In a preferred embodiment, the amount oflight in the output light beam is normalized by the amount of light inthe input light beam, making the result independent of variations in thepower of the light source.

According to another embodiment of the invention, the light source isadapted to generate a polarized input light beam, particularly alinearly polarized input light beam. In a polarized light beam, thevectors of the electrical field (and thus also of the associatedmagnetic field) are not randomly oriented in the plane perpendicular tothe direction of propagation of the light beam but have a regularorientation. This orientation is constant in space for a linearlypolarized light beam and rotates in a regular manner for a circularly orelliptically polarized light beam. Generating an input light beam with apolarization provides it with a characteristic internal feature thataffects the interaction of this beam with other entities, e.g. withoptical components in the light path or with target particles to bedetected. This opens many possibilities that can favorably be exploited,for example the possibility to distinguish light in the output lightbeam that stems from the input light beam from light of other sources,e.g. the ambience.

In a preferred realization of the aforementioned embodiment, the inputlight beam has a linear polarization in the plane of incidence withrespect to an entrance window of the carrier at which the input lightbeam enters the carrier. Additionally or alternatively, the output lightbeam may have a linear polarization in the plane of incidence withrespect to an exit window of the carrier at which the output light beamleaves the carrier. As usual, the “plane of incidence” of a light beamrefers to the plane that comprises said light beam and that isperpendicular to the surface on which said light beam impinges. When alight beam impinges on a surface of the carrier, a (small) fraction ofits light will usually be reflected. Besides the fact that this light islost for other purposes, a particular disadvantage of such a reflectionis that it may disturb other components, e.g. the light detector or alaser in the light source. It is therefore desirable to reduce theamount of light that is reflected at the entrance or exit window of thecarrier. Such a reduction is possible with the proposed setup in whichthe input light beam and/or the output light beam have the explainedpolarization.

In a preferred embodiment of the invention, the entrance window throughwhich the input light beam enters the carrier is placed under Brewsterangle with respect to the input light beam and/or the exit windowthrough which the output light beam leaves the carrier is placed underBrewster angle with respect to the output light beam. As is well knownfrom optics, a reflected beam vanishes if an incident light beam with alinear polarization in the plane of incidence impinges on a surfaceunder the corresponding Brewster angle. If this embodiment is combinedwith the aforementioned one (having a linearly polarized input lightbeam), the reflections at the entrance or exit window of the carrier canbe completely suppressed. The Brewster angle for a particular setup canbe calculated from the fact that, at Brewster angle of incidence, thereis an angle of 90° between the refracted light beam and the direction ofthe (suppressed) reflected light beam.

The invention further relates to a carrier for providing a sample to beinvestigated, wherein said carrier may particularly be suited as acarrier for a microelectronic sensor device of the kind described above.The carrier comprises a sample chamber in which a sample can be providedand which has a transparent inspection wall. The inspection wall has onits interior side a binding surface at which components of a sample cancollect. On its exterior side, the inspection wall has at least oneoptical structure which is designed such that

-   -   (i) an input light beam which is directed from outside the        carrier onto the optical structure enters the inspection wall,    -   (ii) said input light beam is (at least one times) totally        internally reflected in an investigation region at the binding        surface, and    -   (iii) an output light beam comprising at least some of the        totally internally reflected light and/or fluorescence light        emitted by target components at the binding surface leaves the        inspection wall through the optical structure, preferably in a        direction away from the carrier.

The inspection wall will typically have the basic form of a plate with asubstantially parallel interior and exterior surface, wherein theinterior surface comprises the binding surface and wherein the opticalstructure projects outwards from the exterior surface. Moreover, theinspection wall can in principle be any part of the wall of the samplechamber, for example a side wall or the top. Preferably, the inspectionwall is however a part of the bottom of the carrier (or the wholebottom), which has two advantages: First, sample components underlyingsedimentation will concentrate at the binding surface of the bottom.Second, the components of an associated instrument can be disposed belowsaid bottom, thus leaving space at the sides of the carrier for apossible arrangement of further carriers.

The described carrier has the advantage that a sample inside its samplechamber can optically be investigated with an input light beam that istotally internally reflected, thus providing an evanescent field in asmall volume at the binding surface. Effects like absorption orscattering taking place in this small volume will affect the outputlight beam which leaves the carrier. Additionally, fluorescence may bestimulated by the evanescent wave in fluorescent target components andthus provide a further indicator for the target. As both the input lightbeam and the output light beam are directed from the outside towards thecarrier or vice versa, the corresponding light source and light detectorcan be arranged a distance away and separate from the carrier.

The invention further relates to a well-plate which comprises aplurality of carriers of the kind described above, i.e. a plurality ofsample chambers with transparent inspection walls having on theirinterior side a binding surface and on their exterior side at least oneoptical structure, wherein said optical structure allows an input lightbeam coming from outside the carrier to enter the inspection wall, to betotally internally reflected at the binding surface, and then to leavethe inspection wall as an output light beam that is directed away fromthe carrier.

The well-plate combines a plurality of the carriers described above inan array and thus allows a parallel investigation of a multitude ofsamples and/or of one sample in a multitude of investigation assays. Asthe well-plate is based on the described carrier, reference is made tothe above description for more details on the advantages, features andimprovements of said well-plate.

In the following various embodiments of the invention will be describedthat can be applied to a microelectronic sensor device, a carrier and awell-plate of the kind described above.

While it is in principle possible that the carrier has some dedicatedstructure with multiple components of different materials, it ispreferred that the carrier is homogenously fabricated from a transparentmaterial, for example a transparent plastic. The carrier can thusreadily be produced for example by injection moulding.

The investigation region of the carrier has preferably a low roughnessin order to minimize unwanted influences on the (frustrated) totalinternal reflection. With λ being a characteristic (e.g. peak oraverage) wavelength of the light constituting the input light beam, theroughness of the investigation region is preferably less than 0.5λ, mostpreferably less than 0.1λ (which means that the height differencebetween microscopic “valleys” and “tips” of the carrier surface in theinvestigation region is smaller than these values).

The investigation region of the carrier may optionally be covered withat least one type of capture element that can bind one or more targetcomponents. A typical example of such a capture element is an antibodyto which corresponding antigens can specifically bind. By providing theinvestigation region with capture elements that are specific to certaintarget components, it is possible to selectively enrich these targetcomponents in the investigation region. Moreover, undesired targetcomponents can be removed from the binding surface by suitable (e.g.magnetic) repelling forces (that do not break the bindings betweendesired target components and capture elements). The binding surface maypreferably be provided with several types of capture elements that arespecific for different target components. In a microelectronic sensordevice with a plurality of investigation regions, there are preferablyat least two investigation regions having different capture elementssuch that these regions are specific for different target components.

According to another embodiment of the invention, the surface of thecarrier is substantially perpendicular to the input light beam and/or tothe output light beam at the entrance window or exit window where thisbeam enters or leaves the carrier, respectively, i.e. the angle ofincidence lies in a range of about ±5° around 90°. In this case thedirection of the input light beam and/or the output light beam will notor only minimally change during the transition from a surrounding mediuminto the carrier or vice versa. Moreover, reflection will be minimized.Additionally or alternatively, the corresponding regions may also havean anti-reflection coating. To prevent optical feedback into the lightsource (e.g. a laser), it may be preferable to have the incident beam(at most) a few degrees off-perpendicular.

The carrier may particularly comprise at least one surface with a formsimilar or identical to a hemisphere or a truncated pyramid. As will bediscussed in more detail with reference to the Figures, these formsfunction like lenses and/or prisms and thus provide a favorable guidanceof the incident and the output light beam.

The carrier may further optionally comprise a cavity in which a(magnetic or electrical) field generator can at least partially bedisposed. The source of the field can thus be positioned as close aspossible to the binding surface, allowing to generate high fieldstrengths in the investigation region with minimal effort (e.g.electrical currents) and with minimal disturbances for other regions(e.g. neighboring investigation regions). Moreover, such a cavity can beused to center the carrier with respect to the field generator, thelight source and the light detector.

While the microelectronic sensor device may in principle be constructedas a “one-piece” unit of solidly mounted components, it is preferredthat the carrier is designed as an exchangeable component of the device,for example a well-plate. Thus it may be used as a low-cost disposablepart, which is particularly useful if it comes into contact withbiological samples and/or if its coating (e.g. with antibodies) is usedup during one measurement process.

The invention further relates to a method for the detection of targetcomponents comprising label particles, wherein said method comprises thefollowing steps:

-   -   a) Collecting target components at the binding surface of a        carrier.    -   b) Directing an input light beam into the carrier such that it        is totally internally reflected in an investigation region at        the binding surface.    -   c) Determining the amount of light in an output light beam which        comprises at least some of the totally internally reflected        light of the input light beam; preferably the output light beam        comprises only such totally internally reflected light.

The method comprises in general form the steps that can be executed witha microelectronic sensor device of the kind described above. Therefore,reference is made to the preceding description for more information onthe details, advantages and improvements of that method.

In an embodiment of the method, the label particles are manipulated by amagnetic and/or an electrical field, wherein this manipulation mayparticularly comprise the attraction of the particles to or theirrepulsion from the investigation region.

In another embodiment of the method, the amount of light in the inputlight beam is measured and related to the measured amount of light inthe output light beam. Thus variations in the intensity of the inputlight beam can be detected and used to e.g. correct the measured amountof light in the output light beam, making the result independent ofinput light fluctuations.

These and other aspects of the invention will be apparent from andelucidated with reference to the embodiment(s) described hereinafter.These embodiments will be described by way of example with the help ofthe accompanying drawings in which:

FIG. 1 schematically shows the general setup of a microelectronic sensordevice according to the present invention;

FIG. 2 shows the angles of incidence when the input light beam and theoutput light beam are oriented under Brewster angle;

FIG. 3 shows a microelectronic sensor device with a well having aspherical bottom;

FIG. 4 shows the design of FIG. 3 with additional means for focusing alight beam;

FIG. 5 shows a well having a plurality of hemispheres at the bottom;

FIG. 6 shows a well having a bottom in the form of a truncated pyramid;

FIG. 7 shows the design of FIG. 6 with a cavity for an electromagnet;

FIG. 8 is a diagram showing a normalized measurement signal s over timet for solutions with different concentrations of morphine labeled withmagnetic particles;

FIG. 9 is a diagram showing a normalized measurement signal s over timet for solutions containing morphine labeled with magnetic particles anddifferent concentrations of free morphine;

FIG. 10 illustrates the formation of pillars of magnetic beads in amagnetic field;

FIG. 11 is a diagram showing a normalized measurement signal s over timet for solutions with different concentrations of morphine labeled withmagnetic particles when only one step of magnetic attraction is applied;

FIG. 12 is a diagram showing a normalized measurement signal s over timet for solutions containing saliva and morphine labeled with magneticparticles;

FIG. 13 is a diagram showing a normalized measurement signal s over timet for a two-step PTH assay in comparison to a solution containing noPTH;

FIG. 14 shows a dose-response curve for PTH in buffer for opticaldetection;

FIG. 15 is a diagram like that of FIG. 13 for different concentrationsof PTH;

FIG. 16 shows a dose-response curve for PTH in buffer and in blood foroptical detection;

FIG. 17 shows a bead-response curve for detection with a GMR sensor;

FIG. 18 shows a bead-response curve for optical detection.

Like reference numbers or numbers differing by integer multiples of 100refer in the Figures to identical or similar components.

FIG. 1 shows the general setup of a microelectronic sensor deviceaccording to the present invention. A central component of this deviceis the carrier 11 that may for example be made from glass or transparentplastic like poly-styrene. The carrier 11 is located next to a samplechamber 2 in which a sample fluid with target components to be detected(e.g. drugs, antibodies, DNA, etc.) can be provided. The sample furthercomprises magnetic particles 1, for example superparamagnetic beads,wherein these particles 1 are usually bound as labels to theaforementioned target components (for simplicity only the magneticparticles 1 are shown in the Figure).

The interface between the carrier 11 and the sample chamber 2 is formedby a surface called “binding surface” 12. This binding surface 12 mayoptionally be coated with capture elements, e.g. antibodies, which canspecifically bind the target components.

The sensor device comprises a magnetic field generator 41, for examplean electromagnet with a coil and a core, for controllably generating amagnetic field B at the binding surface 12 and in the adjacent space ofthe sample chamber 2. With the help of this magnetic field B, themagnetic particles 1 can be manipulated, i.e. be magnetized andparticularly be moved (if magnetic fields with gradients are used). Thusit is for example possible to attract magnetic particles 1 to thebinding surface 12 in order to accelerate the binding of the associatedtarget component to said surface.

The sensor device further comprises a light source 21, for example alaser or an LED, that generates an input light beam L1 which istransmitted into the carrier 11. The input light beam L1 arrives at thebinding surface 12 at an angle larger than the critical angle θ_(c) oftotal internal reflection (TIR) and is therefore totally internallyreflected as an “output light beam” L2. The output light beam L2 leavesthe carrier 11 through another surface and is detected by a lightdetector 31, e.g. a photodiode. The light detector 31 determines theamount of light of the output light beam L2 (e.g. expressed by the lightintensity of this light beam in the whole spectrum or a certain part ofthe spectrum). The measurement results are evaluated and optionallymonitored over an observation period by an evaluation and recordingmodule 32 that is coupled to the detector 31.

In the light source 21, a commercial DVD (λ=658 nm) laser-diode can beused. A collimator lens may be used to make the input light beam L1parallel, and a pinhole 23 of e.g. 0.5 mm may be used to reduce the beamdiameter. For accurate measurements, a highly stable light source isrequired. However, even with a perfectly stable power source,temperature changes in the laser can cause drifting and random changesin the output.

To address this issue, the light source may optionally have anintegrated input light monitoring diode 22 for measuring the outputlevel of the laser. The (low-pass filtered) output of the monitoringsensor 22 can then be coupled to the evaluation module 32, which candivide the (low-pass filtered) optical signal from the detector 31 bythe output of the monitoring sensor 22. For an improved signal-to-noiseratio, the resulting signal may be time-averaged. The divisioneliminates the effect of laser output fluctuations due to powervariations (no stabilized power source needed) as well as temperaturedrift (no precautions like Peltier elements needed).

A further improvement can be achieved if not (or not only) the laseroutput itself is measured, but the final output of the light source 21.As FIG. 1 coarsely illustrates, only a fraction of the laser outputexits the pinhole 23. Only this fraction will be used for the actualmeasurement in the carrier 11, and is therefore the most direct sourcesignal. Obviously, this fraction is related to the output of the laser,as determined by e.g. the integrated monitor diode 22, but will beaffected by any mechanical change or instability in the light path (alaser beam profile is approximately elliptical with a Gaussian profile,i.e. quite non-uniform). Thus, it is advantageous to measure the amountof light of the input light beam L1 after the pinhole 23 and/or aftereventual other optical components of the light source 21. This can bedone in a number of ways, for example:

-   -   a parallel glass plate 24 can be placed under 45° or a beam        splitter cube (e.g. 90% transmission, 10% reflection) can be        inserted into the light path behind the pinhole 23 to deflect a        small fraction of the light beam towards a separate input-light        monitoring sensor 22;    -   a small mirror at the edge of the pinhole 23 or the input light        beam L1 can be used to deflect a small part of the beam towards        a detector.

The Figure shows a “second light detector” 31′ that can alternatively oradditionally be used to detect fluorescence light emitted by fluorescentparticles 1 which were stimulated by the evanescent wave of the inputlight beam L1. As this fluorescence light is usually emittedisotropically to all sides, the second detector 31′ can in principle bedisposed anywhere, e.g. also above the binding surface 12. Moreover, itis of course possible to use the detector 31, too, for the sampling offluorescence light, wherein the latter may for example spectrally bediscriminated from reflected light L2.

The described microelectronic sensor device applies optical means forthe detection of magnetic particles 1 and the target components forwhich detection is actually of interest. For eliminating or at leastminimizing the influence of background (e.g. of the sample fluid, suchas saliva, blood, etc.), the detection technique should besurface-specific. This is achieved by using the principle of frustratedtotal internal reflection which is explained in the following.

According to Snell's law of refraction, the angles θ_(A) and θ_(B) withrespect to the normal of an interface between two media A and B satisfythe equation

n_(A) sin θ_(A)=n_(B) sin θ_(B)

with n_(A), n_(B) being the refractive indices in medium A and B,respectively. A ray of light in a medium A with high refractive index(e.g. glass with n_(A)=2) will for example refract away from the normalunder an angle θ_(B) at the interface with a medium B with lowerrefractive index such as air (n_(B)=1) or water (n_(B)≈1.3). A part ofthe incident light will be reflected at the interface, with the sameangle as the angle θ_(A) of incidence. When the angle θ_(A) of incidenceis gradually increased, the angle θ_(B) of refraction will increaseuntil it reaches 90°. The corresponding angle of incidence is called thecritical angle, θ_(c), and is given by sin θ_(c)=n_(B)/n_(A). At largerangles of incidence, all light will be reflected inside medium A(glass), hence the name “total internal reflection”. However, very closeto the interface between medium A (glass) and medium B (air or water),an evanescent wave is formed in medium B, which decays exponentiallyaway from the surface. The field amplitude as function of the distance zfrom the surface can be expressed as:

exp(−k√{square root over (n_(A) ² sin²(θ_(A))−n _(B) ²)}·z)

with k=2π/λ, θ_(A) being the incident angle of the totally reflectedbeam, and n_(A) and n_(B) the refractive indices of the respectiveassociated media.

For a typical value of the wavelength λ, e.g. λ=650 nm, and n_(A)=1.53and n_(B)=1.33, the field amplitude has declined to exp(−1)≈0.37 of itsoriginal value after a distance z of about 228 nm. When this evanescentwave interacts with another medium like the magnetic particles 1 in thesetup of FIG. 1, part of the incident light will be coupled into thesample fluid (this is called “frustrated total internal reflection”),and the reflected intensity will be reduced (while the reflectedintensity will be 100% for a clean interface and no interaction).Depending on the amount of disturbance, i.e. the amount of magneticbeads on or very near (within about 200 nm) to the binding surface 12(not in the rest of the sample chamber 2), the reflected intensity willdrop accordingly. This intensity drop is a direct measure for the amountof bonded magnetic beads 1, and therefore for the concentration oftarget molecules. When the mentioned interaction distance of theevanescent wave of about 200 nm is compared with the typical dimensionsof anti-bodies, target molecules and magnetic beads, it is clear thatthe influence of the background will be minimal. Larger wavelengths λwill increase the interaction distance, but the influence of thebackground liquid will still be very small.

The described procedure is independent of applied magnetic fields. Thisallows real-time optical monitoring of preparation, measurement andwashing steps. The monitored signals can also be used to control themeasurement or the individual process steps.

For the materials of a typical application, medium A of the carrier 11can be glass and/or some transparent plastic with a typical refractiveindex of 1.52. Medium B in the sample chamber 2 will be water-based andhave a refractive index close to 1.3. This corresponds to a criticalangle θ_(c) of 60°. An angle of incidence of 70° is therefore apractical choice to allow fluid media with a somewhat larger refractiveindex (assuming n_(A)=1.52, n_(B) is allowed up to a maximum of 1.43).Higher values of n_(B) would require a larger n_(A) and/or larger anglesof incidence.

Advantages of the described optical read-out combined with magneticlabels for actuation are the following:

-   -   Cheap cartridge: The carrier cartridge 11 can consist of a        relatively simple, injection-molded piece of polymer material        that may also contain fluidic channels.    -   Large multiplexing possibilities for multi-analyte testing: The        binding surface 12 in a disposable cartridge can be optically        scanned over a large area. Alternatively, large-area imaging is        possible allowing a large detection array. Such an array        (located on an optical transparent surface) can be made by e.g.        ink jet printing of different binding molecules on the optical        surface. The method also enables high-throughput testing in        well-plates by using multiple beams and multiple detectors and        multiple actuation magnets (either mechanically moved or        electro-magnetically actuated).    -   Actuation and sensing are orthogonal: Magnetic actuation of the        magnetic particles (by large magnetic fields and magnetic field        gradients) does not influence the sensing process. The optical        method therefore allows a continuous monitoring of the signal        during actuation. This provides a lot of insights into the assay        process and it allows easy kinetic detection methods based on        signal slopes.    -   The system is really surface sensitive due to the exponentially        decreasing evanescent field.    -   Easy interface: No electrical interconnect between cartridge and        reader is necessary. An optical window is the only requirement        to probe the cartridge. A contact-less read-out can therefore be        performed.    -   Low-noise read-out is possible.

FIG. 2 illustrates in more detail the angles of incidence of the inputlight beam L1 and the output light beam L2 at the entrance window 14,the binding surface 12, and the exit window 15 of a carrier 11. When theentrance and exit windows 14, 15 are orthogonal to the incoming beam,normally a part of the light (typically around 4%) is reflected back,causing e.g. in the light source 21 non-desirable output fluctuations ofa laser (called “laser feedback”). This distorts the measurement.Furthermore, interference effects can occur in the light detector at thedetection side as well, as typically a perpendicular orientation is usedhere, too, and a coherent light source (laser) is used.

Due to unwanted heating of the measurement cartridge, which occurs e.g.due to the heating of the actuation magnets 41 during operation or dueto other external factors, a slight shift of the positions of thecarrier's facets can lead to slow variations of the intensity, on boththe light source and the detection branch of the setup, that aredifficult to eliminate from the measurement. By placing the lightdetector at an angle (rather than perpendicular) with respect to theincoming output light beam L2, some of the problems occurring at thedetection side of the setup can already be eliminated effectively. Atthe light source side, however, this is not possible.

It is therefore desirable to make the entrance and exit windows 14, 15of the carrier 11 such that reflections are eliminated without the useof expensive optical anti-reflex coatings.

To solve this problem, it is proposed to place the entrance and exitwindows 14, 15 under Brewster angle with respect to the incoming lightbeam, and to provide this beam with a (linear) polarization in the planeof incidence (called “p-polarization”). As is known from optics (cf.e.g. Pedrotti & Pedrotti, Introduction to Optics, Prentice Hall), areflected beam vanishes if the p-polarized incident beam hits thesurface of a (transparent) medium under Brewster angle.

A p-polarized input light beam L1 can be achieved by choosing the rightorientation of a semiconductor laser in the light source, or by using ahalf wave plate to rotate the polarization to the correct orientation.

The propagation of the input light beam L1 inside the carrier 11 isfixed due to the fact that it should impinge on the binding surface 12with an angle θ₃ larger than the critical angle θ_(c) of TIR. This fixesalso the orientation of the entrance and exit windows or facets of thecarrier 11, if their angle θ₂ or θ₆, respectively, with the refractedbeam shall correspond to the Brewster angle. This in turn fixes thedirection of the input light beam L1 and the output light beam L2.

The angle of incidence θ₁ of the input light beam L1 is equal toBrewster angle when the sum of this angle and the angle θ₂ of refractionis 90°. This condition in combination with Snell's law leads to thefollowing formula for the angle of incidence at Brewster angle:

tan(θ₁)=n ₂ /n ₁,

where n₁ is the refractive index of the medium in which the input lightbeam L1 propagates before refraction, normally air, and n₂ of the mediumwhere the refracted ray propagates, normally the plastic of the carrier(e.g. polycarbonate, zeonex or polystyrene).

For the angle θ₂ of the refracted beam one finds

tan(θ₂)=n ₁ /n ₂.

Another condition that needs to be satisfied is that the input lightbeam L1 should be incident at an angle θ₃ close to but beyond thecritical angle θ_(c) of total internal reflection at the binding surface12, i.e.

θ₃>θ_(c) with sin(θ_(c))=n ₃ /n ₂,

where n₃ is the refractive index of the medium above the binding surface12. Furthermore, the angles at the side of the output light beam L2 aremirrored with respect to the input side, i.e.

θ₄=θ₃, θ₅=θ₂, and θ₆=θ₁.

For typical values of n₁=1 (air), n₂=1.5 (transparent plastic), andn₃=1.3 (water like), the following figures can be derived: θ₃=θ₄>60°,θ₁=θ₆=56°, θ₂=θ₅=34°.

By placing the entrance and exit windows of the carrier at Brewsterangle, unwanted reflections back into the laser are prevented withoutthe need of an expensive anti-reflection coating. Furthermore, byplacing the detector at an angle, rather than perpendicular,interference effects on the detector side can be prevented as well. Bydoing so, expansion or shrinkage of the carrier/cartridge during ameasurement, e.g. due to thermal effects, will not influence themeasurement result.

In the environment of a laboratory, well-plates are typically used thatcomprise an array of many sample chambers (“wells”) in which differenttests can take place in parallel. FIGS. 3-7 show different possibleembodiments of one well of such a well-plate that are particularlysuited for an application of the explained measurement principle. Theproduction of these (disposable) wells is very simple and cheap as asingle injection-moulding step is sufficient.

The light source 121 shown in FIG. 3 is arranged to produce a parallellight beam L1, incident at the well bottom surface at an angle largerthan the critical angle θ_(c). To prevent excess reflection of thisinput light beam L1 at the first interface from air to the carrier 111(e.g. glass or plastic material), the bottom of the well comprises ahemispherical shape 114 of radius R, with its centre coinciding with thedetection surface 112. The input light beam L1 is directed towards thissame centre. At the reflection side, a photodetector such as aphotodiode 131 is positioned to detect the intensity of the output lightbeam L2. A typical diameter D of the well 102 ranges from 1 to 8 nm. TheFigure further indicates a magnet 141 for generating magnetic actuationfields inside the well 102 (this magnet is not shown in the followingFigures for simplicity).

FIG. 4 shows an alternative embodiment in which the light sourcecomprises some optical element like a lens 222 to produce an input lightbeam L1 which is substantially focused to the centre of the hemisphere214. At the detection side, a similar optical element 232 can be used tocollect and detect the light intensity of the output light beam L2.

In a further development of the measuring procedure, multiple inputlight beams and output light beams can be used to simultaneously detectthe presence of different target molecules at different locations in thesame well. FIG. 5 shows in this respect a well with multiple hemispheres314 a, 314 b on the well bottom that can be used to couple the lightfrom multiple input light beams L1 a, L1 b to respective investigationregions 313 a, 313 b on the bottom of the well. Multiple photodetectors(not shown) may be used in this case to measure the multiple outputlight beams L2 a, L2 b.

FIG. 6 shows an alternative embodiment in which a prism or truncatedpyramidal structure 414 is used to couple the light of the input lightbeam L1 and the output light beam L2. The sloped edges of the pyramidshould be substantially perpendicular to these light rays. Advantages ofthis design are that it is simple to produce and does not block beamsfrom neighboring areas. Neighboring wells are indicated in this Figureby dashed lines.

As indicated in FIG. 6, it is possible to use a single, parallel inputlight beam L1 with a diameter covering all detection areas on the wellbottom. As a detector, multiple photodiodes can be used, aligned witheach individual detection area. Alternatively, a CCD or CMOS chip (notshown) such as used in a digital camera can be used to image thereflected intensity response of the entire well bottom, including alldetection areas. Using appropriate signal processing, all signals can bederived as with the separate detectors, but without the need for prioralignment.

FIG. 7 shows a further embodiment in which the well bottom 511 comprisesan open cavity 515 with its center outside the optical path of the inputlight beam(s) L1 and the output light beam(s) L2. This allows thefollowing advantageous features:

-   -   A (T-shaped) ferrite core 542 of a magnetic coil 541 for        improved field intensity and concentration can be placed close        to the binding surface 512, allowing a compact and low-power        design.    -   A self-aligning structure is achieved: if the optics and the        magnetic field generator 541 are fixed, an auto-alignment of the        well on the ferrite core 542 takes place.

The magnetic beads 1 that are used in the described embodiments of theinvention are typically poly-styrene spheres filled with small magneticgrains (e.g. of iron-oxide). This causes the beads to besuper-paramagnetic. The refractive index of poly-styrene is nicelymatched to the refractive index of a typical substrate material ofwell-plates. In this way optical outcoupling of light is enhanced.

Experimental Results A

In the following, some experimental results will be described that wereobtained in a setup with a well-plate like that of FIG. 3. Standard 96wells polystyrene titerplates were used with a flat bottom (6 mm indiameter, about 1 mm bottom thickness). To get the hemispherical bottom,glass lenses were attached to the bottom using refractive index matchedimmersion oil (n=1.55). The glass lenses were polished down from ahemispherical shape (6 mm diameter) to a thickness of 2 mm. The modelassay chosen for the set of experiments is drugs of abuse in saliva.Drugs of abuse are generally small molecules that only possess oneepitope and for this reason cannot be detected by a sandwich assay. Acompetitive or inhibition assay is the method to detect these molecules.A well-known competitive assay setup is to couple the target moleculesof interest onto a surface, and link antibodies to a detection tag (e.g.enzyme, fluorophore, or magnetic particle). This system was used toperform a competitive assay between the target molecules from the sampleand the target molecules on the surface, using the tagged antibodies.The tag in these experiments was a magnetic particle. Upon actuation, apermanent magnet was placed under the well by mechanical movement. Thedistance between the bottom of the well and the magnet was about 2 mm. Apermanent magnet in the well was used for magnetic washing.

FIG. 8 shows the normalized measurement signal s over time t for a firstsensitivity test. For that, the bottom of a well was prepared fordetection of the target molecules. The target under investigation wasmorphine. Morphine is a small molecule, with only one epitope, so acompetitive assay has to be performed to indicate the amount of morphinein a sample. A clear polystyrene surface (96 wells titerplate) wascoated for 2 hrs with a range of concentrations of BSA-morphine from 1pg/ml to 1 μg/ml. Then functionalized superparamagnetic nanoparticles“MP” (300 nm Carboxyl-Adembeads functionalized with monoclonal antimorphine antibodies) solved in PBS+10 mg/ml BSA+0.65% Tween-20 wereinserted into the wells (1:20 dilution of MPs, total amount of solutionwas 50 μl). The MPs were attracted to the surface by alternatedapplication of magnetic forces (in the order of 10 fN) as indicated bysymbol A in FIG. 8. In the end, unbound particles were removed from thesurface by a washing step, indicated by symbol W in FIG. 8. The Figureshows that the lowest concentration of BSA-morphine (10 pg/ml) yieldsthe largest dynamic measurement range. Also, the steepness of the curveafter actuation is the largest, enabling fast response/short measuringtime and the highest sensitivity.

To test the sensitivity of the assay, the ability of free morphine tocompete for functionalized MP binding to the surface was tested. FIG. 9shows the resulting normalized signal s collected by the detector as afunction of time t. A clear polystyrene surface (96 wells titerplate)was coated for 2 hrs with 10 pg/ml BSA-morphine. MPs functionalized withanti-morphine antibodies premixed with a defined amount of free morphinesolved in PBS+10 mg/ml BSA+0.65% Tween-20 were inserted into the wells(1:20 dilution of MPs, total amount of solution was 40 μl). As describedabove and indicated in the Figure, the MPs were actuated four times att=30 s, t=140 s, t=210 s, t=290 s during 15 sec (cf. symbol A). At t=390s the non-bound MPs have been removed from the well by means of magneticwashing W, i.e. the non-bound MPs are removed by applying a magneticforce using a permanent magnet in the fluid above the binding surface.

It can be seen from the Figure that for the highest concentrations offree morphine, the signal reduction (after magnetic washing W) is low,while for a low concentration of free morphine the signal reduction ishigh (a high concentration of MPs on the surface leading to a clearreduction in signal after magnetic washing W).

The signal reduction during actuation and magnetic relaxation as foundin these experiments, together with information already collected frommicroscopic investigations proposes the following interpretation of theresults: Upon magnetic actuation, MPs are concentrated to the surface,without showing an increase in binding to the surface (no signaldecrease). Upon removing of the magnetic field, the signal dropsindicating MPs binding to the surface. Application of a magnetic fieldthen induces pillar formation: MPs become magnetized and those freelymovable (a-specifically bound MPs and MPs freely in solution) will bindto the specific bound MPs in the direction of the magnetic field lines,which are perpendicular to the binding surface. This state isillustrated in FIG. 10, which also indicates the evanescent field EF.Since the evanescence detection system will only detect MPs at thesurface, pillar formation during magnetic actuation will result in areduction in signal change. Upon removal of the magnetic field, the MPswill lose their magnetic property, and fall to the surface again wherebinding can take place.

To obtain a fast assay, the actuation scheme can be optimized using theabove results. FIG. 11 shows a dose-response curve on polystyrene wellscoated with 10 pg/ml BSA-morphine. MPs functionalized with anti-morphineantibodies premixed with a defined amount of free morphine solved inPBS+10 mg/ml BSA+0.65% Tween-20 were inserted into the wells (1:20dilution of MPs, total amount of solution was 40 μl, final morphineconcentrations between 1 and 1000 ng/ml). MPs were actuated at A using apermanent magnet below the well for 15 seconds, to up-concentrate theMPs near the surface. Next, the MPs were allowed to bind to the surfacefor 60 seconds. The data show that already after 20 seconds the bindingrate of magnetic particles to the surface is a direct measure for theconcentration of free morphine in solution. This means that themeasurement procedure can be simplified and more rapid, since no washingstep is needed. For this to occur rapidly, the magnetic up-concentrationstep A is necessary.

Next, the background signal from saliva was tested. Filtered saliva wasintroduced in a well and the signal was followed for 120 seconds. Thebackground is negligible as can be seen in FIG. 12. As a comparison, thesignal of MPs in PBS+10 mg/ml BSA+0.65% Tween20 mixed with 0.1 ng/mlmorphine is included as well. At t=13 s, both the saliva (SL) and themorphine solution+MPs were injected. It can be seen that the backgroundsignal from the saliva is <1% and can be neglected.

Experimental Results B

To verify the sensitivity of the detection method a two-step PTH(PTH=parathyroid hormone) assay was carried out in the describedwell-plates on an optical substrate. In FIG. 13 the signal transients s(arbitrary units) are plotted as function of time t for both a blanc (0nM, upper curve) and a relatively high (4 nM) concentration. A cleardifference in the kinetic binding regime is observed and also a clearsignal difference after washing W remains.

In order to compare magnetic read-out (via Giant Magneto-Resistance(GMR) sensors as they are for example described in the WO 2005/010543 A1or WO 2005/010542 A2) with optical read-out (via the principle offrustrated total internal reflection explained above), the PTH doseresponse curve is plotted in FIG. 14.

The corresponding transient curves for the optically detected PTH assayare given in FIG. 15. The dose-response curve in FIG. 14 is measured ina buffer matrix. The curve shows the optical signal s as percentage ofthe signal caused by reflection from an empty substrate. It isinteresting to note that the curve is linear on a log-log scale (similarto the magnetically detected curve). Furthermore, detection limits canbe calculated according to ‘blanc+2*standard deviation of the blanc’(wherein “blanc” indicates the signal level when testing a sample withzero target concentration).

For the magnetic read-out this value is equal to 3 pM. For the opticalexperiment, which was done with a very basic experimental set-up, thisvalue was equal to 13 pM. It can be concluded that both detectiontechniques seem to have the same sensitivity.

Next it is very important to verify the background signal for theoptical detection method when measuring in complex matrices. For thisreason the same PTH assay was carried out in a blood matrix. From theresults shown in FIG. 16 (Bld.=blood, Buf.=buffer) it is clear that theresulting dose-response very well matches the curve that was measured inbuffer. Also the blank signal is very low. This nice property isattributed to the fact that the total internal reflection is caused bythe refractive index difference between the optical substrate materialand the matrix. The matrix can consist of different components such asplasma, (red blood) cells, etc. However, all these components have asignificantly lower refractive index than the substrate material.Therefore, total internal reflection is not influenced by the matrix.Only when beads are bound (e.g. high-index polystyrene with magneticgrains) the total internal reflection is frustrated and a drop inreflected intensity can be measured.

Experimental Results C

An important proof for the proposed technology is the so-calledbead-response curve. It gives an indication of the signal change perbead attached to the sensor surface. Ideally, detection of a single beadis possible (in presence of noise, disturbances). In this situationfurther improving the detection technology is not needed anymore. Thebiological detection limit can then only be improved by methods such asupconcentration of beads (in a catch-assay), etc. FIG. 17 shows thebead-response curve in case of detection with a GMR-type of sensor and300 nm beads (Δs=signal change; BD=bead density; NB=number of beads).For these beads the detection limit was 3 beads on 40 μm² for a samplingfrequency of 1 Hz.

In order to estimate the bead-response for optical detection, a numberof samples (glass-slides) were prepared with various beadconcentrations. The resulting surface coverage was determined using anoptical microscope, followed by a measurement of the optical signal(change) s compared to a clean reference sample without beads. Theexperimental data as obtained with a simple set-up are plotted in FIG.18. In this set-up, the noise level corresponds to a signal change at asurface coverage SC of 0.01%. These data show that the sensitivity ofthis technique is at least similar to state-of-the-art results using GMRsensors at the same bead concentrations.

In summary, the invention relates to a microelectronic sensor device forthe detection of target components that comprise label particles, forexample magnetic particles 1. The sensor device comprises a carrier 11with a binding surface 12 at which target components can collect andoptionally bind to specific capture elements. An input light beam L1 istransmitted into the carrier and totally internally reflected at thebinding surface 12. The amount of light in the output light beam L2 isthen detected by a light detector 31. Evanescent light generated duringthe total internal reflection interacts with the label particles 1 boundto target particles at the binding surface 12 leading to absorptionand/or scattering and will therefore be missing in the output light beamL2. This can be used to determine the amount of target components at thebinding surface 12 from the amount of light in the output light beam L2,L2 a, L2 b. A magnetic field generator 41 is optionally used to generatea magnetic field B at the binding surface 12 by which the magnetic labelparticles 1 can be manipulated, for example attracted or repelled.

The label particles 1 are for instance magnetic beads, which meansmagnetic particles MP, in an example paramagnetic, ferromagnetic, orsuper-paramagnetic particles or beads. These label particles 1 aresubject to reflection of the light beam L1 impinging at the bindingsurface 12 of the carrier. The more label particles 1 are bound to thebinding surface 12 the more the light beam L1 will not be totallyinternal reflected at the binding surface 12 but an evanescent wave willbe generated. The light beam L2 reflected by the binding surface 12 isso called frustrated by the effect of scattering of the incoming lightbeam L1 by the label particles 1. The more label particles 1 bound tothe binding surface the more the reflected light beam L2 is frustrated.The light detector 31, 131 measures the light beam L2 coming from thebinding surface 12 and uses the reflected light beam L2 for measuringthe amount of label particles 1 bound at the binding surface 12. Themore label particles 1 bound to the binding surface 12 the morescattering of the light beam L1 due to the label particles 1 takes placewith the generation of the evanescent wave. The label particles 1enabling the effect described have for instance the following features.Magnetic beads of a width of roughly 300 nm of uniform superparamagneticparticles containing a polymer core shell structure. These labelparticles 1 or magnetic beads show a scatter of light beam L1 which issufficient for detection of the reflected light beam L2 to determine thelabel particles 1. Label particles 1 of similar materials and widths arealso feasible, for instance label particles 1 of a width of 200 nm.Nevertheless, the scatter of light from only the target components towhich the label particles 1 bind is found not to be suitable fordetection. This means the direct measurement of target components byfrustrated internal reflection to measure the amount of these targetcomponents is not possible. The detection and subsequent calculation ofthe amount of target particles 1 is made possible by the scattering oflight at the label particles 1. Herewith, no detection of fluorescenceof fluorescent material is needed, as used in state of the art withother optical detection systems. Further in this context it is to bestressed that detection of fluorescence light emitted by the targetcomponents is an additional feature which is combinable with thedescribed method and sensor device.

While the invention was described above with reference to particularembodiments, various modifications and extensions are possible, forexample:

-   -   In addition to molecular assays, also larger moieties can be        detected with sensor devices according to the invention, e.g.        cells, viruses, or fractions of cells or viruses, tissue        extract, etc.    -   The detection can occur with or without scanning of the sensor        element with respect to the sensor surface.    -   Measurement data can be derived as an end-point measurement, as        well as by recording signals kinetically or intermittently.    -   The particles serving as labels can be detected directly by the        sensing method. As well, the particles can be further processed        prior to detection. An example of further processing is that        materials are added or that the (bio)chemical or physical        properties of the label are modified to facilitate detection.    -   The device and method can be used with several biochemical assay        types, e.g. binding/unbinding assay, sandwich assay, competition        assay, displacement assay, enzymatic assay, etc. It is        especially suitable for DNA detection because large scale        multiplexing is easily possible and different oligos can be        spotted via ink jet printing on the optical substrate.    -   The device and method are suited for sensor multiplexing (i.e.        the parallel use of different sensors and sensor surfaces),        label multiplexing (i.e. the parallel use of different types of        labels) and chamber multiplexing (i.e. the parallel use of        different reaction chambers).    -   The device and method can be used as rapid, robust, and easy to        use point-of-care biosensors for small sample volumes. The        reaction chamber can be a disposable item to be used with a        compact reader, containing the one or more field generating        means and one or more detection means. Also, the device, methods        and systems of the present invention can be used in automated        high-throughput testing. In this case, the reaction chamber is        e.g. a well-plate or cuvette, fitting into an automated        instrument.

Finally it is pointed out that in the present application the term“comprising” does not exclude other elements or steps, that “a” or “an”does not exclude a plurality, and that a single processor or other unitmay fulfill the functions of several means. The invention resides ineach and every novel characteristic feature and each and everycombination of characteristic features. Moreover, reference signs in theclaims shall not be construed as limiting their scope.

1. A microelectronic sensor device for the detection of targetcomponents comprising label-particles (1), comprising a) a carrier (11,111, 211, 311, 411, 511) with a binding surface (12, 112, 512) at whichtarget components can collect; b) a light source (21, 121) for emittingan input light beam (L1, L1 a, L1 b) into the carrier such that it istotally internally reflected in an investigation region (13, 313 a, 313b) at the binding surface; c) a light detector (31, 131) for determiningthe amount of light in an output light beam (L2, L2 a, L2 b) thatcomprises at least some of the totally internally reflected light. 2.The microelectronic sensor device according to claim 1, wherein thetotally internally reflected light beam becomes frustrated resulting ina decrease of the totally internally reflected light intensity when thelabel-particles (1) are macroscopic scattering and/or absorbingparticles.
 3. The microelectronic sensor device according to claim 1,wherein the microelectronic sensor device comprises a field generator(41, 141, 541) for generating a magnetic field (B) and/or an electricalfield that can affect the label particles (1).
 4. The microelectronicsensor device according to claim 1, wherein the microelectronic sensordevice comprises a sample chamber (2, 102) adjacent to the bindingsurface (12, 112, 512) in which a sample with target components can beprovided.
 5. The microelectronic sensor device according to claim 1,wherein the microelectronic sensor device comprises an evaluation module(32) for determining the amount of target components in theinvestigation region (13, 313 a, 313 b) from the measured output lightbeam (L2, L2 a, L2 b).
 6. The microelectronic sensor device according toclaim 1, wherein the microelectronic sensor device comprises a recordingmodule (32) for monitoring the determined amount of reflected light overan observation period.
 7. The microelectronic sensor device according toclaim 1, wherein the carrier comprises a plurality of investigationregions (313 a, 313 b) at which different input light beams (L1 a, L1 b)can be totally internally reflected.
 8. The microelectronic sensordevice according to claim 7, wherein the microelectronic sensor devicecomprises a scanning module for optically coupling the light sourceand/or the light detector to different investigation regions (313 a, 313b) at the binding surface.
 9. The microelectronic sensor deviceaccording to claim 7, wherein the microelectronic sensor devicecomprises a plurality of light sources and/or a plurality of lightdetectors that are optically coupled to different investigation regions(313 a, 313 b) at the binding surface (12).
 10. The microelectronicsensor device according to claim 3, wherein the microelectronic sensordevice comprises a plurality of individually controllable fieldgenerators (41, 141, 541) associated to different investigation regions.11. The microelectronic sensor device according to claim 1, wherein themicroelectronic sensor device comprises label particles (1) with amantle of a transparent material, particular a material with a similarrefractive index as the carrier (11, 111, 211, 311, 411, 511).
 12. Themicroelectronic sensor device according to claim 1, wherein themicroelectronic sensor device comprises a second light detector (31′)for determining fluorescence light emitted by target components at thebinding surface.
 13. The microelectronic sensor device according toclaim 1, wherein the microelectronic sensor device comprises aninput-light monitoring sensor (22, 22′) for determining the amount oflight in the input light beam (L1).
 14. The microelectronic sensordevice according to claim 13, wherein the input-light monitoring sensor(22, 22′) is disposed inside the light source (21) or outside from it.15. The microelectronic sensor device according to claim 13, wherein themicroelectronic sensor device comprises an evaluation module (32) forrelating the determined amount of light in the output light beam (L2) tothe determined amount of light in the input light beam (L1).
 16. Themicroelectronic sensor device according to claim 1, wherein the lightsource (21) is adapted to generate a polarized input light beam (L1),particularly a linearly polarized input light beam (L1).
 17. Themicroelectronic sensor device according to claim 16, wherein the inputlight beam (L1) has a linear polarization (p) in the plane of incidencewith respect to an entrance window (14) at which it enters the carrier(11) and/or that the output light beam (L2) has a linear polarization inthe plane of incidence with respect to an exit window (15) at which itleaves the carrier (11).
 18. The microelectronic sensor device accordingto claim 1, wherein the entrance window (14) at which the input lightbeam (L1) enters the carrier (11) is under Brewster angle (θ₁) withrespect to the input light beam (L1) and/or that the exit window (15) atwhich the output light beam (L2) leaves the carrier (11) is underBrewster angle (θ₅) with respect to the output light beam (L2).
 19. Acarrier (11, 111, 211, 311, 411, 511) for a sample to be investigated,the carrier comprising a sample chamber (2, 102) with a transparentinspection wall that has on its interior side a binding surface (12,112, 512) and on its exterior side at least one optical structure (114,214, 314 a, 314 b, 414), wherein the optical structure is designed suchthat an input light beam (L1, L1 a, L1 b) directed from outside thecarrier onto the optical structure enters the inspection wall and istotally internally reflected in an investigation region (13, 313 a, 313b) at the binding surface, and that an output light beam (L2, L2 a, L2b) and/or fluorescence light emitted by target components at the bindingsurface leaves the inspection wall through the optical structure.
 20. Awell-plate comprising a plurality of carriers according to claim
 19. 21.The carrier of claim 19, wherein the carrier (11, 111, 211, 311, 411,511) consists of a transparent material.
 22. The carrier of claim 19,wherein the investigation region (13, 313 a, 313 b) has a low roughness.23. The carrier of claim 19, wherein the investigation region (13, 313a, 313 b) is covered with at least one capture element that can bindtarget components.
 24. The carrier according to claim 19 wherein thesurface of the carrier (11, 111, 211, 311, 411, 511) is substantiallyperpendicular to the input light beam (L1, L1 a, L1 b) and/or the outputlight beam (L2, L2 a, L2 b) at the entrance window (14) or exit window(15) where this beam enters or leaves the carrier, respectively.
 25. Thecarrier of claim 19, wherein the carrier (11, 111, 211, 311, 411, 511)comprises at least one surface part with a form like a hemisphere (114,214, 314 a, 314 b) or a truncated pyramid (414).
 26. The carrier ofclaim 19, wherein the carrier (511) comprises a cavity (515) in which afield generator (541) can at least partially be disposed.
 27. Thecarrier of claim 19, wherein the carrier (11, 111, 211, 311, 411, 511)is designed as an exchangeable component.
 28. A method for the detectionof target components comprising label particles (1), comprising a)collecting the target components at a binding surface (12, 112, 512) ofa carrier (11, 111, 211, 311, 411, 511); b) directing an input lightbeam (L1, L1 a, L1 b) into the carrier such that it is totallyinternally reflected in an investigation region (13, 313 a, 313 b) atthe binding surface (12, 112, 512); c) determining the amount of lightof an output light beam (L2, L2 a, L2 b) that comprises at least some ofthe totally internally reflected light.
 29. The method according toclaim 28, wherein the label particles (1) are manipulated by a magneticor electrical field (B), particularly attracted or repelled from theinvestigation region (13, 313 a, 313 b).
 30. The method according toclaim 29, wherein the amount of light in the input light beam (L1) ismeasured and related to the measured amount of light in the output lightbeam (L2).
 31. The carrier of claim 22, wherein the investigation region(13, 313 a, 313 b) has a roughness of 0.5λ or less, with λ being acharacteristic wavelength of the input light beam (L1).
 32. The carrierof claim 22, wherein the investigation region (13, 313 a, 313 b) has aroughness of 0.1λ or less, with λ being a characteristic wavelength ofthe input light beam (L1).